1. Field of the Invention
The present invention relates generally to cell lines and particularly to methods and compositions for the development of cell lines which exhibit less heterogeneity in their asparagine-linked glycans, with most such glycans being produced in the form of Man.sub.3 GlcNAc.sub.2.
2. Description of the Related Art
It is now well established that many important cell surface proteins and secretory proteins produced by eukaryotic cells contain one or several carbohydrate groups, and are thus said to be "glycosylated" (for a general overview see, e.g., Darnell et al., 1986). These carbohydrates serve many possible functions. For example, the addition of carbohydrate groups to a protein can affect the conformation of a polypeptide, making it more resistant to enzymatic degradation, or increase its solubility, due to the many hydroxyl groups present on carbohydrates. Further, some carbohydrate residues are important in directing the particular glycosylated protein to a particular location within the cell.
Importantly, carbohydrate residues have been found to play a potentially profound role in the pharmacology of various glycosylated proteins produced by cells, such as in the case of human tissue plasminogen activator (t-PA) and human erythropoietin (EPO) (Spellman et al., 1989; Takeuchi et al., 1990). Interestingly, it has been discovered that t-PA exists in two forms, depending on the presence (type I) or absence (type II) of carbohydrate at position Asn-184, with type II exhibiting a specific activity of about 30-50% greater than that of type I t-PA (Spellman et al., 1989). Similarly, in the case of EPO it was found that sequential removal of galactose and N-acetylglucosamine from the outer chain moieties of the desialylated Asn-type sugar chains raised the activity of the hormone up to four to five times that of intact EPO (Takeuchi et al., 1990).
The cellular mechanisms which underlie carbohydrate attachment to proteins is complex. In eukaryotes, sugars are commonly attached to one of four different amino acid residues and are classified as either O-linked (serine or threonine) or N-linked (asparagine). The structures of N- and O-linked oligosaccharides are very different, and different sugar moieties are found to be attached in each type. For example, in the case of N-linked oligosaccharides, the sugar residues are synthesized from a common precursor and subsequently processed. The precursor is a branched oligosaccharide containing three glucose, nine mannose and two N-acetylglucosamine molecules (GlcNAc) that is linked by a pyrophosphate residue to dolichol, a long-chain unsaturated lipid. The mannose residues are attached to the free amino group of asparagine by means of the two residues of GlcNAc.
The assembly process involves at least 16 enzymatic reactions to generate oligosaccharides attached to dolichol in pyrophosphate linkage and containing 2 residues of N-acetylglucosamine, 9 residues of mannose, and 3 residues of glucose (Kornfeld et al., 1985). This precursor is then transferred in its entirety to the amino group of asparagine through the action of oligosaccharide transferase inside the lumen of the rough endoplasmic reticulum. However, due to the action of various cellular enzymes, much heterogeneity is found to exist in the N-linked glycan, such as in terms of the number of mannose residues that are attached in "high mannose" structures, and in terms of the number, length, and terminal sugars of the branches of the "complex-type" structures.
Due to the importance of carbohydrate residues on the biological and physiological parameters of proteins, particularly the N-linked glycans, much research has been conducted in elucidating the mechanisms involved in glycosylation. In particular, various cell lines having one or more defects in their glycosylation mechanisms have been generated both as an aid I0 to understanding the underlying mechanism, and in attempts to provide cells which produce less heterogenic glycosylation patterns in proteins having N-linked glycans. The generation of proteins having less heterogeneity in the N-linked glycan structure is a particularly important goal, in that heterogeneity in the N-linked glycan is undesirable, such as in production and purification of useful glycoproteins (reviewed in Stanley, 1989), in biophysical studies of glycoproteins, or where one desires to modify the biological activity of the glycoprotein (see, e.g., Takeuchi et al., 1990).
Unfortunately, past attempts to develop cell lines which produce N-linked glycans that are smaller and less heterogeneous have met with only limited success. For example, in the C.sub.6 /36 mosquito cell line, which does not synthesize complex-type oligosaccharides, only about half of the N-linked glycans had the structure Man.sub.3 GlcNAc.sub.2, with the remainder having heterogeneous compositions with 5 to 9 mannose residues (Hsieh et al., 1984). Presumably, the latter fraction resulted from incomplete trimming of precursors containing 9 mannose residues. A CHO line carrying the Lec1 and Lec6 mutations (Stanley, 1984; Hunt, 1980) was shown to produce glucosylated Man.sub.7 GlcNAc.sub.2 -P-P-dolichol which eventually gave rise to N-linked glycans with 4 mannose residues due to defective N-acetylglucosaminyltransferase I. However, there was also significant accumulation of oligosaccharides with 5-7 mannose residues, presumably due to incomplete trimming. Several other CHO lines have also been described carrying the Lec1 mutation in combination with others mutations (Stanley, 1989), but the smallest N-linked glycan produced by these cells had 5 mannose residues.
Accordingly, there is currently a need for methodology for the development of cell lines which are capable of producing N-linked glycans having a reduced heterogeneity. More specifically, there is a need for the development of cell lines that have the ability to produce N-linked glycans having greater proportions of glycans existing in the form of simplified structures such as Man.sub.3 GlcNAc.sub.2. In particular, there is a need for cell lines which can be used as recombinant host cells for the production or recombinant glycoproteins having simplified glycosylation patterns, which may provide means of altering the underlying biologic parameters of the produced protein or improving the yield or purity of isolation.